Lipid membranes are the physical barriers and functional interfaces between cellular compartments, and are therefore involved in nearly every aspect of cellular physiology. To accommodate this exquisite functionality, membranes are composed of a vast array of lipids and proteins that self-organize on a variety of length and time scales. Disturbances to the lipid constituents of cellular membranes are associated with diverse disease phenotypes, including cardiovascular disease, autoimmunity, osteoporosis, neurological disorders, and cancer. Moreover, unique to lipids among other cellular macromolecules, lipid phenotypes can be affected directly by dietary lipid abundance and composition. Most notable are the deleterious health consequences associated with overconsumption of saturated and trans-unsaturated fats, and, conversely, the plethora of beneficial effects of ?-3 polyunsaturated fats (PUFA). Despite the broad significance of membrane phenotypes, the detailed lipid compositions determining these phenotypes, lipidomic remodeling during cellular processes, and the susceptibility of membrane composition and function to dietary lipid inputs have not been widely characterized. Recent analytic breakthroughs in holistic quantitation of membrane lipids have revealed that cells produce hundreds of distinct lipid species, and that cellular lipidomes are remarkably plastic, capable of rapid, large-scale, functional rearrangements. These observations prompt the hypothesis that both cell autonomous and exogenous factors drive membrane lipidome remodeling and that the resulting membrane phenotypes directly regulate cell function. To test this broad hypothesis, we propose a combination of mass spectrometry, membrane biophysics, and stem cell biology to characterize lipidomic and biophysical membrane remodeling during the differentiation of mesenchymal stem cells (MSCs), and how dietary lipids affect this remodeling to regulate MSC differentiation. In Aim 1, we will define the compositional and biophysical differentiation of whole membranes and isolated plasma membranes as MSCs undergo differentiation into adipocytes and osteoblasts. These observations will be combined with bioinformatic approaches to uncover the compositional determinants of physical properties in biological membranes. In Aim 2, we will extend these observations by investigating the modulation of membrane composition and physical properties by dietary lipids - specifically cholesterol, saturated / trans-unsaturated fats, and ?-3 PUFA. Further, we will evaluate the functional aspects of membrane remodeling by investigating the consequences of lipidomic disturbances on lineage-specific MSC differentiation. Finally, we will explore the molecular mechanisms behind these observations by the experiments proposed in Aim 3. We will dissect the mitogenic and differentiation pathways regulated by membrane remodeling to identify the molecules responsible for transducing membrane phenotypes into cellular signals. The long-term goal of this line of research is to identify modulators of membrane phenotypes for treatment of diseases associated with lipidomic perturbations and for promotion of desired cellular phenotypes, e.g. osteogenic differentiation of MSCs in osteoporosis.